本研究は、2022年度JST基礎研究(日本移植学会)の支援を受けて行われました。

この実験計画書は、京都大学の動物実験委員会で承認されました。この研究では、市販の供給源から入手した、10週間齢以上、体重25gから30gのC57BL/6マウスを利用しました( 「材料の表」を参照)。すべての動物に2.5%イソフルランを麻酔し(施設で承認されたプロトコルに従って)、特定の病原体を含まない条件下で維持し、すべての実験手順は京都大学の動物実験規則に準拠して実施されました。研究に使用された適切な機器は、 材料表 にリストされ、 図1 と 図2に示されています。
1.動物の選択
<ol><li>体重25〜30 gのマウスを使用してください。 注: マウス肝臓同種移植片2 は、主要な組織適合遺伝子複合体障壁を越えて一般に受け入れられていますが、この研究では、寒冷虚血再灌流障害10 の詳細なメカニズムに焦点を当てるために、同系 MOLT モデルを選択しました。体重が25g未満のマウスは、薄い胆管にインナーステントを挿入できないため、推奨されません。同様に、体重が30 gを超えるマウスは、血管の周囲に大量の腹腔内脂肪が存在するため、推奨されません。</li>
</ol>2.袖口作り
<ol><li>PVとIHIVC用にそれぞれ20Gと16Gの静脈カテーテルを準備します。</li>
	<li>メスを使ってカテーテルを切ってカフを作ります。袖口の本体の長さは2 mm、延長ハンドルは1 mmである必要があります(図3A)。 注意: ハンドルが大きすぎると、袖口の挿入が困難になる場合があります。</li>
	<li>袖口の表面に、メスの刃の裏側を使用して糸を固定するための浅い溝を作成します。 注意: メスの刃の裏側を袖口に当てるときは、刃の先端が約60度の角度になっていることを確認してください( 図3Bを参照)。最初の溝を作ったら、メスの刃を固定し、袖口を回転させます。理想的には2つの溝が推奨されますが、1つの溝でも問題なく十分です。</li>
</ol>3.袖口アタッチメント
<ol><li>プラスチック製の長方形の容器に、小さな角氷を置き、その上に小さな金属製のカップ(直径6cm)を置きます。カップに臓器保存液( 材料の表を参照)を入れ、その中に同系移植片を置きます。</li>
	<li>綿棒を使用して同系肝移植片(別のドナーマウスから抽出)を穏やかに転がし、肝門が上を向くようにします。IVCの内腔に溜まった血液を臓器保存液で完全に除去します。</li>
	<li>たとえば、脾臓静脈に取り付けられた糸を使用して、PVを袖口に通します( 図4Aを参照)。</li>
	<li>カフハンドルを12時の位置にして、ハンドルとカフの両方をブルドッグclで固定しますamp ( 材料の表を参照)、PVの端から2〜3mmの位置に配置します。</li>
	<li>大きな血管鉗子と固定型を使用してブルドッグクランプをさらに固定し、術野を確立します( 図4Bを参照)。</li>
	<li>PVルーメンを約15〜20倍の倍率で注意深く検査します。必要に応じて、水を滴下すると、内腔の視認性を高めることができます。</li>
	<li>PVルーメンをそっとつかみ、カフを通して外在化します。</li>
	<li>完全に外部化されたら、8-0 を使用して二重結紮で固定します。袖口の溝に沿った絹糸(図4C、D)。 注:結紮糸が緩んでいないことを確認してください。また、結紮点が大きすぎるとカフ挿入の邪魔になることもあるので注意が必要です。結紮点は任意の方向に配置できます。</li>
	<li>PVに使用したのと同様の手順を使用して、カフをIHIVCに取り付けます(図4E)。 注意: IHIVCをカフで固定するときは、肝実質に噛まないようにしてください。PVよりネックが短いので、クランプ位置を慎重に決めてください。</li>
	<li>IHIVCの基部に糸を通し、IHIVCを一時的に合字させることで、スリップノットを作成します(図5)。この糸は再灌流後に除去されます。</li>
</ol>4. 門脈再建
<ol><li>イソフルランを2.5%で麻酔を誘発し、それを0.6%に減らしてから、公開された文献2に記載されているように、天然の肝臓を抽出します。開腹術の前に、ヨードフォアと70%エタノールを交互に繰り返して手術部位を少なくとも3回消毒します。</li>
	<li>肝葉が適切に配置され、カフ付きPVにねじれやねじれがないことを確認してください。</li>
	<li>ピーン鉗子を使用して受信者のPVエッジをそっとつかみ、万力で固定します。 注:この記事では、万力とは、ペアン鉗子をテーブルに固定するために使用される機械装置を指します。万力の詳細については、 材料の表を参照してください。</li>
	<li>レシピエントのPVに血管クランプを適用し、8-0を渡しますその周りの絹糸。</li>
	<li>PVエッジから約1mmの円周セクションを0.5mm作成します(図6A)。専用の器具(先端を切り取り、L字型に曲げた27Gの針)を使用して、生理食塩水を内腔に通しながら穴を拡大します。</li>
	<li>まっすぐな鉗子を使用してカフハンドルをつかみ、内腔に挿入します。</li>
	<li>完全に挿入したら、カフを8-0で固定します。絹糸。固定には1回の結紮で十分です。</li>
	<li>血管クランプを取り外し、肝臓を再灌流します。.</li>
	<li>ピーン鉗子を慎重に離し、門脈再建を完了します。PVの再構築プロセス全体には約5分かかります。</li>
</ol>5. 下大静脈下再建
<ol><li>肝葉を適切に配置し、レシピエントIHIVC背側に取り付けられたピーン鉗子を移植片の右下葉に動かし、万力で固定します。</li>
	<li>レシピエントのIVIHCの周囲に残っている肝臓組織を、綿棒で優しくこすって取り除きます。</li>
	<li>8-0で合格受信者のIVIHCの周りの絹糸。</li>
	<li>レシピエントのIVIHCエッジから約1mmに4/0.5円周セクションを作成します(図6B)。</li>
	<li>通常の生理食塩水を内腔に導入した後、カフを内腔に挿入します。 注:首が短いため、袖口はPVよりも浅く挿入されることがよくあります。</li>
	<li>8-0で袖口を慎重に固定します絹糸を通し、血管クランプとピーン鉗子を取り外します。</li>
</ol>6.術後ケア
<ol><li>加熱パッドと加温ランプを利用して、術後の回復を促進します。</li>
	<li>血圧、心拍数、呼吸数、体温、および食物と水の消費量を継続的に監視します。</li>
	<li>術後の痛みを軽減するためのカルプロフェンの皮下注射(5 mg / kg、6時間ごとまたは必要になるまで)を投与します。地域の機関のガイドラインに従ってください。 注:マウスの状態が悪化した場合、実験は直ちに中止され、マウスは二酸化炭素の吸入によって安楽死させられました。</li>
	<li>組織サンプルを採取するには、マウスを再麻酔し、IACUC承認の方法で安楽死させます。</li>
</ol>

マウス肝移植における効率的な血管吻合のための戦略

<ol>
	<li>Qian, S. G., Fung, J. J., Demetris, A. V., Ildstad, S. T., Starzl, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+the+mouse.">Orthotopic liver transplantation in the mouse.</a> Transplantation. 52 (3), 562-564 (1991).</li><li>Yokota, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+mouse+liver+transplantation+to+study+liver+biology+and+allograft+tolerance.">Orthotopic mouse liver transplantation to study liver biology and allograft tolerance.</a> Nat Protoc. 11 (7), 1163-1174 (2016).</li><li>Shen, X. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+responses+in+a+new+mouse+model+of+prolonged+hepatic+cold+ischemia+followed+by+arterialized+orthotopic+liver+transplantation.">Inflammatory responses in a new mouse model of prolonged hepatic cold ischemia followed by arterialized orthotopic liver transplantation.</a> Liver Transpl. 11 (10), 1273-1281 (2005).</li><li>Kageyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion+injury+in+allogeneic+liver+transplantation:+A+role+of+CD4+T+cells+in+early+allograft+injury.">Ischemia-reperfusion injury in allogeneic liver transplantation: A role of CD4 T cells in early allograft injury.</a> Transplantation. 105 (9), 1989-1997 (2021).</li><li>Li, T., Hu, Z., Wang, L., Lv, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Details+determining+the+success+in+establishing+a+mouse+orthotopic+liver+transplantation+model.">Details determining the success in establishing a mouse orthotopic liver transplantation model.</a> World J Gastroenterol. 26 (27), 3889-3898 (2020).</li><li>Tian, Y., R&#252;diger, H. A., Jochum, W., Clavien, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+arterialized+and+nonarterialized+orthotopic+liver+transplantation+in+mice:+Prowess+or+relevant+model.">Comparison of arterialized and nonarterialized orthotopic liver transplantation in mice: Prowess or relevant model.</a> Transplantation. 74 (9), 1242-1246 (2002).</li><li>Steger, U., Sawitzki, B., Gassel, A. M., Gassel, H. J., Wood, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+hepatic+rearterialization+on+reperfusion+injury+and+outcome+after+mouse+liver+transplantation.">Impact of hepatic rearterialization on reperfusion injury and outcome after mouse liver transplantation.</a> Transplantation. 76 (2), 327-332 (2003).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+various+techniques+of+mouse+liver+transplantation.">A review of various techniques of mouse liver transplantation.</a> Transplant Proc. 45 (6), 2517-2521 (2013).</li><li>Kojima, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+carcinoembryonic+antigen-related+cell+adhesion+molecule+1-T+cell+immunoglobulin+domain+and+mucin+domain-containing+protein+3+cross+talk+alleviates+liver+transplant+injury+in+mice+and+humans.">T cell carcinoembryonic antigen-related cell adhesion molecule 1-T cell immunoglobulin domain and mucin domain-containing protein 3 cross talk alleviates liver transplant injury in mice and humans.</a> Gastroenterology. 165 (5), 1233-1248 (2023).</li><li>Yokota, S., Yoshida, O., Ono, Y., Geller, D. A., Thomson, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+transplantation+in+the+mouse:+Insights+into+liver+immunobiology,+tissue+injury,+and+allograft+tolerance.">Liver transplantation in the mouse: Insights into liver immunobiology, tissue injury, and allograft tolerance.</a> Liver Transpl. 22 (4), 536-546 (2016).</li></ol>

開示すべき利益相反のある著者はいません。

血管再建を学ぶことは、MOLTを成功させるための最も困難な側面です。カフの品質は、マウスのサイズが小さいことを考えると、再建の難しさに大きく影響します5。この記事では、カフの準備、取り付け、および再建の詳細なプロトコルを提供します。
カフの準備と接続に関する以前のレポートと大きな違いはありませんが、いくつか...

マウス同所性肝移植(MOLT)は、1991年に最初に報告された効果的な実験方法です1。遺伝子改変マウスや各種研究用試薬を用いたこの実験モデルは、温冷虚血や再灌流障害の研究に重要な役割を果たしてきました。しかし、このモデルの高い技術的複雑さは、肝移植のための基礎医学の開発を妨げてきました2。MOLTは、(1)ドナーマウスからの肝臓の回収、(2)バックテーブル手術、(3)レシピエントへの肝臓移植の3つの主要なステップから成ります。これらのレシピエント手順の中で、血管吻合は最大の課題をもたらします。下大静脈上吻合は通常、手縫い2で完了しますが、下肝下大静脈(IHIVC)と門脈(PV)は、手縫いの縫合の代わりにプラスチック製の袖口を使用してより効率的に再建できます。
無肝期は、レシピエントの天然肝臓の除去と移植片の移植片の移植の間の間隔を意味します。一貫した結果を得るには、無肝時間を20分未満に制限することが不可欠です。その結果、このモデルを採用した研究は、特定の機関に限定されている3,4,5,6,7,8,9。MOLTの様々な段階の中で、PVとIHIVCのスムーズな再建を実現することは、無肝時間を最小化し、移植を成功させるために重要です。
PVおよびIVC再建は、手縫い縫合糸2,5,8と比較して、カフ技術が血管吻合を簡素化するため、一般に血管カフを使用して行われます。血管カフの準備とカフの確実な取り付けに関連する技術は、レシピエントの血管再建の複雑さに大きな影響を与えます。私たちの目的は、カフ法に関連する多くの技術的なヒントを詳細に視覚的に指導し、それによって学習曲線を短縮することです。これらのビデオ クリップは、カフを血管に取り付け、レシピエント手術中に PV と IHIVC を再構築する方法を明確に理解します。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16 G intravenous catheter</td>
			<td>TERUMO</td>
			<td>SR-FF2032</td>
			<td>IHIVC cuff</td>
		</tr>
		<tr>
			<td>20 G intravenous catheter</td>
			<td>TERUMO</td>
			<td>SR-FF1651</td>
			<td>PV cuff</td>
		</tr>
		<tr>
			<td>8-0 braid silk</td>
			<td>Natsume Seisakusho</td>
			<td>CR9-80B2</td>
			<td>8-0 silk</td>
		</tr>
		<tr>
			<td>Belzer UW Cold Storage Solution&#160;</td>
			<td>Astellas</td>
			<td></td>
			<td>Organ preservation fluid</td>
		</tr>
		<tr>
			<td>Bulldog clamp</td>
			<td>B BRAUN</td>
			<td>FB329R</td>
			<td>Bulldog clamp</td>
		</tr>
		<tr>
			<td>C57BL/6 mice&#160;&#160;</td>
			<td>Oriental Bio Service</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane inhalation solution</td>
			<td>Viatris</td>
			<td></td>
			<td>Anesthesic</td>
		</tr>
		<tr>
			<td>Micro Blunted Tips 0.1 mm x 0.06 mm&#160;</td>
			<td>F.S.T</td>
			<td>11253-20</td>
			<td>Straight microforceps</td>
		</tr>
		<tr>
			<td>Micro Serrefine Clamp Applicator with Lock</td>
			<td>F.S.T</td>
			<td>18056-14</td>
			<td>Vessel clip applicator</td>
		</tr>
		<tr>
			<td>Micro Serrefines&#160;</td>
			<td>F.S.T</td>
			<td>18055-4</td>
			<td>Vessel clip</td>
		</tr>
		<tr>
			<td>No.11 Spare Blades</td>
			<td>FEATHER Safety Razor</td>
			<td>11</td>
			<td>Blades</td>
		</tr>
		<tr>
			<td>Ophthalmic scissor, round handle</td>
			<td>B BRAUN</td>
			<td>FD103R</td>
			<td>Microscissor</td>
		</tr>
		<tr>
			<td>Plastic rectangular-shaped container&#160;&#160;</td>
			<td>Daiso</td>
			<td></td>
			<td>10 cm long, 15 cm wide and 6 cm high</td>
		</tr>
		<tr>
			<td>SuperGrip Tips</td>
			<td>F.S.T</td>
			<td>00649-11&#160;</td>
			<td>Curved microforceps</td>
		</tr>
		<tr>
			<td>SZX7</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td>Microscope</td>
		</tr>
	</tbody>
</table>

vascular reconstruction with the cuff technique in mouse orthotopic liver transplantation

マウス同所性肝移植は、肝虚血や再灌流障害の根底にあるメカニズムを調べるための有効な方法論です。しかし、技術的な課題は、この貴重な実験モデルを活用し、これらのスキルを次世代に継承する上での障壁となっています。この手順の最も困難な側面は、門脈 (PV)、下肝下大静脈 (IHIVC)、および下大静脈上を含む血管再建です。縫合糸ではなくプラスチック製のカフを使用することで、PVやIHIVCの再建をよりスムーズに行うことができます。静脈カテーテルで作ったカフを移植血管の先端に取り付け、カフをレシピエント血管に介在させることで血管を再構築します。最も重要な2つの側面は、血管の内腔を適切に視覚化することと、過度の力の使用を避けることです。私たちの目的は、レシピエント手術におけるカフ技術を使用した血管再建の技術的概要を提供することです。カフ技術に関するこれらの技術的ヒントは、顕微外科医が血管再建を容易にし、研究を前進させるのに役立つと期待されています。

PV再建は、門脈のクランプを外したときに曲がりくねった状態がなく、肝臓が均一に灌流されている場合に成功します。無肝時間が25分を超えるとマウスの死亡リスクが高まるため、無肝時間は20分未満にする必要があります。IHIVCの再建は、移植片からの血液逆流がない場合、成功したと見なされます。
臓器保存溶液を使用して移植片を低温で1時間保存すると、再灌流?...

Watch this Scientific Journal Video about Author Spotlight: Exploring Cold Ischemia and Warm Reperfusion Injury in Fatty Liver Transplantation at JoVE.com

著者スポットライト:脂肪肝移植における冷虚血と温再灌流障害の探索

このプロトコルは、マウス同所性肝移植におけるカフ技術を使用した血管再建のための技術情報を提供します。

マウス同所性肝移植におけるカフ技術による血管再建

Mouse orthotopic liver transplantation is an effective methodology for investigating the underlying mechanisms of liver ischemia and reperfusion injury. ...

肝移植、特に低温虚血と温再灌流障害の研究に重点を置いています。そのための研究ツールとして、マウス肝移植モデルを用いています。テクニックが難しいため、習得して一貫した結果を達成するには長い時間がかかる場合があります。将来的には、脂肪肝移植の研究も行っていきたいと考えています。これは、臨床肝移植で克服すべき問題です。

vascular-reconstruction-with-cuff-technique-mouse-orthotopic-liver

Research

JoVE Journal

Medicine

Vascular Reconstruction with the Cuff Technique in Mouse Orthotopic Liver Transplantation

1.1K ビュー.  Kyoto University. 肝移植、特に低温虚血と温再灌流障害の研究に重点を置いています。そのための研究ツールとして、マウス肝移植モデルを用いています。テクニックが難しいため、習得して一貫した結果を達成するには長い時間がかかる場合があります。将来的には、脂肪肝移植の研究も行っていきたいと考えています。これは、臨床肝移植で克服すべき問題です。

ビデオ: 著者スポットライト:脂肪肝移植における冷虚血と温再灌流障害の探索

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

ラット同所性肝移植

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

医学

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

血管疾患の研究のためのマウスにおける同所性大動脈移植

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

肝動脈再建を用いた部分同所性肝移植のラットモデルのための外科的処置

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

急性肝障害における流れ調節性門脈動脈血と異補助ラット肝移植

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

マウスで同所後肢移植

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

大動脈弁移植 - 教則ビデオのマウスモデルでスリーブを用いた

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

変更されたカフを用いた膵臓移植マウス モデル

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

同所性同種のモデルラット Suprahepatic 上大静脈再建のため新規磁気吻合法を用いた肝移植

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

マウス頸部異所性心臓移植モデルのための修正カフ法

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...

マウスへの肝移植と同種移植片の開存性の分析

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

著者スポットライト:マウスモデルにおけるMicroCT血管造影による移植研究の強化 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

マイクロコンピュータ断層撮影血管造影を用いたマウスの同所性肝移植の成功

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...